Tailored excitation for Fourier transform ion cyclotron mass spectrometry

Secreted proteins of Mycobacterium tuberculosis are implicated in its disease pathogenesis and so are considered as potential diagnostic and vaccine candidates. The search for these has been slow, even though the entire genome sequence of M. tuberculosis is now available; of the 620 protein spots resolved by 2-D gel electrophoresis, 114 secreted proteins have been identified, but for only 13 has the primary structure been partly characterized. For comparison, in this top down mass spectrometry (MS) approach the secreted proteins were precipitated from cell culture filtrate, resuspended, and examined directly by electrospray ionization (ESI) Fourier transform MS. The ESI spectra of three precipitates showed 93, 535, and 369 molecular weight (M r ) values, for a total of 689 different values. However, only ϳ10% of these values matched (Ϯ1 Da) the DNA predicted M r values, but these identifications were unreliable. Of nine molecular ions characterized by MS/MS, only one protein match was confirmed, and its isotopic molecular ions were overlapped by those of another protein. MS/MS identified a total of ten proteins by sequence tag search, of which three were unidentified previously. The low success of M r matching was due to unusually extensive posttranslational modifications, including loss of a signal sequence, loss of the N-terminal residue, proteolytic degradation, oxidation, and glycosylation. Although in eubacteria the latter is relatively rare, a 9 kDa protein showed 7 hexose attachments and two 20 kDa proteins each had 20 attachments. For MS/MS, electron capture dissociation was especially effective. (J Am Soc Mass Spectrom 2003, 14, 253-261)

Section: Glycoprotein Preparationmentioning

confidence: 99%

Top down characterization of secreted proteins from Mycobacterium tuberculosis by electron capture dissociation mass spectrometry

ElNaggar

Sze

et al. 2003

“…P(OCH 3 ) 2 ϩ was subjected to on-resonance excitation at its cyclotron frequency for about 0.8 -1.2 s while pulsing in helium at a nominal pressure of about 10 Ϫ5 Torr. The transfer was achieved by grounding the conductance limit plate for 85-100 s. The transferred ions were allowed to cool by IR emission [20] and by collisions with Ar present at about 10 Ϫ5 Torr for a period of about 1 s. P(OCH 3 ) 2 ϩ was isolated by using a stored-waveform inverse Fourier transform (SWIFT) [21,22] excitation pulse to eject all unwanted ions, and allowed to react with GV, DG, GFA, AAA, AGG, PGG, GPA, VGG, GGV, N-acetyl-AAA, and PLG-amide desorbed as neutral molecules into that cell by means of LIAD. The number of laser shots was varied between 50 and 200.…”

Section: Instrumentationmentioning

confidence: 99%

Evaluation of a novel approach for peptide sequencing: Laser-induced acoustic desorption combined with P(OCH₃)₂⁺ chemical ionization and collision-activated dissociation in a fourier transform ion cyclotron resonance mass spectrometer

Somuramasami

Kenttämaa

2007

A novel mass spectrometric method has been developed for obtaining sequence information on small peptides. The peptides are desorbed as intact neutral molecules into a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR) by means of laser-induced acoustic desorption (LIAD). Reactions of the neutral peptides with the dimethoxyphosphenium ion, P(OCH 3 ) 2 ϩ , occur predominantly by addition of the peptide to P(OCH 3 ) 2 ϩ followed by the loss of two methanol molecules, thus yielding product ions with the composition (peptide ϩ P Ϫ 2H) ϩ . Upon sustained off-resonance irradiation for collision-activated dissociation (SORI-CAD), the (peptide ϩ P Ϫ 2H) ϩ ions undergo successive losses of CO and NH¢CHR or H 2 O, CO, and NH¢CHR to yield sequence-related fragment ions in addition to the regular a n -and b n -type ions. Under the same conditions, SORI-CAD of the analogous protonated peptides predominantly yields the regular a n -and b n -type ions. The mechanisms of the reactions of peptides with P(OCH 3 ) 2 ϩ and the dissociation of the (peptide ϩ P Ϫ 2H) ϩ ions were examined by using model peptides and molecular orbital calculations. (J Am Soc Mass Spectrom 2007, 18, 525-540)

“…The multiply-charged ligand:receptor complexes [M ϩ L ϩ 11H ϩ ], were isolated by the SWIFT technique [21,22]. A Synrad carbon dioxide IR laser (SYNRAD Inc., Mukilteo, WA) beam was used to dissociate each noncovalent complex to release its bound ligand.…”

Section: Swift Isolation and Laser-induced Ligand Releasementioning

confidence: 99%

“…The complexes of interest were first isolated using the SWIFT technique [21,22]. This technique ejects from the Penning ion trap all ions having m/z different from that of the complex of interest (Figure 2) Figure 3, (bottom).…”

Section: Non-covalent Bindingmentioning

confidence: 99%

“…The non-covalent complexes between this SH2 domain and the preferred phosphopeptide ligands were identified by use of the ESI technique to generate ions, FT-ICR MS joined with the stored-waveform inverse Fourier transform (SWIFT) technique to trap and isolate the non-covalent complex, and subsequent IRMPD-irradiation to release the preferred binding ligands [21,22]. We then compare and discuss the gas-phase results in terms of those derived from conventional screening assays.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Fourier transform-ion cyclotron resonance mass spectrometric resolution, identification, and screening of non-covalent complexes of Hck Src homology 2 domain receptor and ligands from a 324-member peptide combinatorial library

Wigger

Eyler

Benner

et al. 2002

Self Cite

The preferred ligands for the Hck Src homology 2 domain among a combinatorial library containing 324 different peptides were determined in a single experiment involving Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS), electrospray ionization (ESI), stored-waveform inverse Fourier transformation (SWIFT), and infrared multiphoton laser disassociation (IRMPD). These were compared with the results obtained by conventional screening of the peptide library in solution using affinity chromatography. The results reported here show that by combining ESI, FT-ICR MS, SWIFT, and IRMPD, ligands likely to bind under physiological conditions are rapidly and efficiently identified, even from complex library mixtures. In the gas phase some discrimination against hydrophobic ligands could be observed. However, the illustrated feasibility of identifying high affinity ligand via gas-phase screening of complex library mixtures should lead to broad applications in the development of ligands for proteins with interesting biological activity, the first step that must be taken to develop a therapeutic agent. Important in the application of FT-ICR methods to biological macromolecules are "soft" ionization techniques, which generate macromolecular ions without fragmentation. Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) are two of these [4,5]. ESI involves spraying an aqueous solution of a biological macromolecule under "quasiphysiological" conditions directly into a mass spectrometer. The solvent evaporates in the vacuum from the droplets, gently "elevating" a biological ion from its native environment in solution. This process frequently leaves non-covalent macromolecular complexes intact and allows the mass spectrometric observation of enzyme-substrate, receptor-ligand, peptide-peptide, protein-subunit, oligonucleotide-toxin/drug, and DNA duplex binding in the gas phase [6].In many cases, binding constants estimated from mass spectrometric studies match the binding constants obtained in solution to within an order of magnitude [7]. The gentleness of ESI allows H/D exchange experiments in the gas phase to give information about protein conformation and protein/protein interactions based on measurements of the exchange rates of protein backbone amide protons [8,9]. This combination of